Mutant polypeptides and uses thereof

A polypeptide comprising an amino acid sequence with at least 90% amino acid sequence homology to SEQ ID NO:1, wherein said amino acid sequence comprises one to five mutations at the following X positions of SEQ ID NO:1:

• R1-95X96R97-98X99R100-122X123R124-186X187R188-203X204R205-211X212R213-272X273X274X275R276-323X324R325-327X328R329-R359X360R361-365X366R367-381X382R383-398, wherein:
• X382 is mutated to a different amino acid selected from W and equivalent amino acids, optionally F382W:
• X328 is mutated to a different amino acid selected from V and equivalent amino acids, optionally L328V;
• X187 is mutated to a different amino acid selected from M and equivalent amino acids, optionally I187M;
• X96 is mutated to a different amino acid selected from L and equivalent amino acids;
• X99 is mutated to a different amino acid selected from L and equivalent amino acids;
• X123 is mutated to a different amino acid selected from I and equivalent amino acids;
• X204 is mutated to a different amino acid selected from I and equivalent amino acids;
• X212 is mutated to a different amino acid selected from F, Y, and equivalent amino acids;
• X273 is mutated to a different amino acid selected from C and equivalent amino acids;
• X274 is mutated to a different amino acid selected from F and equivalent amino acids;
• X275 is mutated to a different amino acid selected from I and equivalent amino acids;
• X324 is mutated to a different amino acid selected from L, E, and equivalent amino acids;
• X360 is mutated to a different amino acid selected from Y and equivalent amino acids;
• X366 is mutated to a different amino acid selected from V, C, G, and equivalent amino acids;
• and each R is the same as the corresponding amino acid in SEQ ID NO:1;
• and optionally, wherein the amino acid sequence comprises at least one mutation selected from F382W, L328V, and I187M, preferably all three, more preferably comprising the amino acid sequence of SEQ ID NO:462 or SEQ ID NO:462 without the periplasmic tag.

A method of identifying a substrate or an inhibitor of a LDH, comprising any one or more of the steps of:

• (a) obtaining a crystal, or the coordinates of a crystal, of a polypeptide comprising SEQ ID NO:5, wherein the crystal is in space group P2(1) with cell parameters a=133.18 Å, b=110.83 Å, c=162.20 Å;
• (b) obtaining or determining the three-dimensional structure of said polypeptide using the crystal of (a) by an X-ray diffraction method;
• (c) displaying the three dimensional structure of said complex on a performing computer by inputting said crystal structure data of said polypeptide, wherein the performing computer comprises a computer program to generate said three dimensional structure and to identify a substrate or an inhibitor;
• and (d) selecting a substrate or an inhibitor of the active site of the polypeptide.

A method for designing a LDH substrate or an inhibitor, the method comprising any one or more of the steps of:

• (a) obtaining a crystal, or the coordinates of a crystal, in space group P2(1) with cell parameters a=133.18 Å, b=110.83 Å, c=162.20 Å, of a complex consisting of a polypeptide of SEQ ID NO:5 bound to the substrate or inhibitor at its binding location;
• (b) obtaining or determining the three dimensional structure of the complex using the crystal obtained in (a) by an X-ray diffraction method to obtain the atomic coordinates of the structure;
• (c) providing on a computer the atomic coordinates of the three dimensional structure of the complex;
• and (d) utilizing a program operated by the computer to design a chemical compound predicted to bind to the polypeptide of SEQ ID NO:5 at the substrate or inhibitor's binding location and either act as a substrate or inhibit the LDH, based on said three dimensional structure.

A method of identifying a compound that binds the polypeptide of SEQ ID NO:5, comprising:

• (a) obtaining a crystal comprising a protein consisting of SEQ ID NO:
• 5 in space group P2(1) with cell parameters a=133.18 Å, b=110.83 Å, c=162.20 Å;
• (b) determining the three-dimensional structure of said polypeptide by X-ray diffraction to obtain the atomic coordinates of Appendix I;
• (c) contacting the polypeptide structure defined by the atomic coordinates of Appendix I, or a subset thereof with a test compound;
• and (d) detecting an interaction between the compound and the atomic coordinates, wherein an energetically favored interaction between the test compound and the atomic coordinates is indicative of a compound that binds said polypeptide.

A method of identifying a substrate or an inhibitor of a LDH, comprising any one or more of the steps of:

• (a) obtaining a crystal, or the coordinates of a crystal, of a polypeptide comprising SEQ ID NO:
• 9, wherein the crystal is in space group space group P2(1) with cell parameters a=88.7 Å, b=111.22 Å, c=120.42 Å;
• (b) obtaining or determining the three-dimensional structure of said polypeptide using the crystal of (a) by an X-ray diffraction method;
• (c) displaying the three dimensional structure of said complex on a performing computer by inputting said crystal structure data of said polypeptide, wherein the performing computer comprises a computer program to generate said three dimensional structure and to identify a substrate or an inhibitor;
• and (d) selecting a substrate or an inhibitor of the active site of the polypeptide.

A method for designing a LDH substrate or an inhibitor, the method comprising any one or more of the steps of:

• (a) obtaining a crystal, or the coordinates of a crystal, in space group P2(1) with cell parameters a=88.7 Å, b=111.22 Å, c=120.42 Å, of a complex consisting of a polypeptide of SEQ ID NO:
• 9 bound to the substrate or inhibitor at its binding location;
• (b) obtaining or determining the three dimensional structure of the complex using the crystal obtained in (a) by an X-ray diffraction method to obtain the atomic coordinates of the structure;
• (c) providing on a computer the atomic coordinates of the three dimensional structure of the complex;
• and (d) utilizing a program operated by the computer to design a chemical compound predicted to bind to the polypeptide of SEQ ID NO:
• 9 at the substrate or inhibitor's binding location and either act as a substrate or inhibit the LDH, based on said three dimensional structure.

A method of identifying a compound that binds the polypeptide of SEQ ID NO:

• 9, comprising:
• (a) obtaining a crystal comprising a protein consisting of SEQ ID NO:
• 9 wherein the crystal is in space group space group P2(1) with cell parameters a=88.7 Å, b=111.22 Å, c=120.42 Å;
• (b) determining the three-dimensional structure of said polypeptide by X-ray diffraction to obtain the atomic coordinates of Appendix 2;
• (c) contacting the polypeptide structure defined by the atomic coordinates of Appendix 2, or a subset thereof with a test compound;
• and (d) detecting an interaction between the compound and the atomic coordinates, wherein an energetically favored interaction between the test compound and the atomic coordinates is indicative of a compound that binds said polypeptide.